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Received: 22-Mar-2015; Revised: 11-May-2015; Accepted: 11-May-2015
This article has been accepted for publication and undergone full peer review but has not been through the copyediting,
typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of
Record. Please cite this article as doi: 10.1002/elps.201500153.
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Electrode Substrate Innovation for Electrochemical
Detection in Microchip Electrophoresis
Edward P. Randviir and Craig E. Banks*
Faculty of Science and Engineering, School of Chemistry and the Environment,
Division of Chemistry and Environmental Science, Manchester Metropolitan University,
Chester Street, Manchester M1 5GD, Lancs, UK
Submission to: Electrophoresis
*To whom correspondence should be addressed.
Email: [email protected]; Tel: ++(0)1612471196; Fax: ++(0)1612476831
Website: www.craigbanksresearch.com
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Abstract
Microchip Electrophoresis (ME) represents the next generation of miniaturised
electrophoretic devices and carry benefits such as significant improvement in analysis times,
lower consumption of reagents and samples, flexibility, and procedural simplicity. The
devices provide a separation method for complex sample matrices and an on-board detection
method for the analytical determination of a target compound. The detection part of ME is
increasingly leaning towards electrochemical methods, thus the selectivity and sensitivity of
detection in ME is dependent upon the chosen working electrode composition in addition to
operating conditions of the chip such as separation voltage. Given the current plethora of
electrode materials that are available, there exists a possibility to creatively integrate
electrodes into ME. This review will overview the application of several electrode materials,
from the old through to the new. A particular recent focus has been the selectivity element of
MEs overcome with the use of enzymes, carbon composites, and screen-printed technologies.
Introduction
One problem with the real world application of analytical techniques lies within the
sample matrix that target analytes arrive within. For example, clinical samples are contained
within blood, urine, or saliva, while environmental samples could be anything from mud,
water, air, rock, sludge, or sands. The complex nature of such matrices leads to a high
possibility of interference or noise from a detection system, thus a host of analytical
procedures are utilised to process a sample, such as acid digestion, liquid or solid phase
extractions, or even electrophoretic separation, prior to the actual measurement step in order
to isolate the intended chemical species one wishes to observe. This multi-phase protocol can
be time consuming and requires samples to be transported to a lab, when ideally the injection,
separation, and measurement would be all done at the point of sample collection.
The aim of Microchip Electrophoresis (ME) is to scale specific lab techniques (such
as Capillary Electrophoresis, CE) into one portable handheld unit with a centimetre-sized
geometry, therefore integrating multiple sample handling processes a combine with some
measurement steps [1]. The separation part of ME was focussed on in detail prior to 2005,
with the technology maturing quite rapidly [1]. However scale-down detection strategies for
such technologies unfortunately lagged behind. Many chips still required the use of Laser-
Induced Fluorescence (LIF) or Mass Spectrometry (MS) for the detection of target species,
both of which require large off-chip measurement systems. Ideally one would combine both
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the separation and detection elements into ME, therefore the external use of LIF or MS is
contradictory to this philosophy. Research has since been directed towards the development
of a detection strategy that can be fabricated into a chip, thus completing the design of a self-
contained microsystem when combined with electrophoretic separation and injection.
Electrochemistry has thus attracted considerable attention as a chemical detection method in
ME devices [2, 3] due to the possibility of on-chip integration using electrodeposited or
printed circuitry, in addition to its relative inexpensiveness, speed of measurement, and
portability benefits when compared to LIF or MS.
The purpose of this opening review is to assess electrode substrates that are
commonly implemented for the purpose of electrochemical detection in ME devices and
demonstrate how researchers have developed new methods to incorporate electrodes into
microchips. The review is designed to focus primarily upon potentiostatic types of
electrochemical detection modes using controlled potential techniques such as those often
implemented for electroanalytical applications. The review discusses specific examples of
electrode substrates applied for the detection or target analytes, thus the reader requires a
good understanding of electrochemical redox reactions for complete understanding of the
work discussed within.
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Microchip Electrophoresis
In order to explain how an ME chip can function, we make reference to a chip created
for the detection of creatinine; a Schematic is given in Figure 1 [4]. The device utilises CE
and conductivity measurements to separate and detect creatinine within a sample of human
serum. Serum samples are inserted at point 1 and subject to a high voltage injection step,
allowing the matrix to migrate into the device. The potential for injection is applied using
electrodes at points A and B. The charged species migrate down channel 3 towards the
separation channel in the chip at point 4. Electrodes C and D apply a secondary electric field
that allows separation of the target analyte. It is at this point that the target species is
physically removed/isolated from the sample matrix, which in theory allows ease of
detection. The electrodes at point 6 detect the separated chemical species, in this case using
conductivity. The conductivity of the sample is plotted as a function of time in an
electropherogram. In this approach the retention time of the target species in required to be
known, unless of course the working electrode is tailored to only transduce responses for
specified targets. As in all electrochemical measurements, a conductivity measurement would
be arbitrary without some reference value for the conductivity measurement to be compared
against. Therefore, point E allows for conductivity measurements of the sample without prior
separation. The conductivity of the separated analyte is thus standardised against the
conductivity of the bulk solution in this case. If we assume that ME devices generally operate
using similar protocols (high voltage injection followed by CE separation), we can focus
specifically upon the detection aspect of the chips, as depicted by point 6 in Figure 1. The
detection and reference electrodes must be tailored specifically for different applications and
has been the focus of much work since the birth of ME devices, particularly in recent times.
The remainder of the review focusses upon a short selection of integrated electrodes utilised
for ME devices and strategies employed to design, produce, and improve working electrodes
for ME purposes.
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Working Electrodes for Microchip Electrophoresis
Many ME systems utilise working electrodes engineered for a specific target analyte.
CE electrodes, such as platinum, copper, or silver chloride electrodes are designed to separate
matrices by their m/z ratios [5], but this review focusses upon working electrodes that are
designed to detect the target species under scrutiny. A working electrode in this review is
defined as an electrode that is responsible for measuring the concentration of an intended
target species. Potentiostatic detection electrodes in ME devices generally employ
amperometric systems, since there is a wide acceptance that such methods are acceptable for
detecting analytes contained in liquid-based effluents [1]. Yet, voltammetric measurements
are equally possible. This section discusses some of the first electrodes implemented for
microfluidic electrochemical detection purposes, and proceeds to discuss the progression of
electrode development from the 1990s to the present day.
Early Devices
The first reports of ME working electrodes date back to 1996 by Gavin and Ewing
[6]. Their photolithographic platinum electrodes were embedded into a chip constructed from
quartz. An amperometric method was demonstrated to detect dopamine using channel flow
electrophoresis with electrochemical detection; something that was previously problematic
due to the electric fields imposed in capillary electrophoresis affecting electrochemical
results. The chip was designed in a manner that allowed for less electric field interference
from injection and separation, by etching the electrodes in a recess some 25 µm from the
height of the channel flow. This way the electric field in the capillary is less likely to interfere
with the dynamic electrochemistry that takes place at the electrode surfaces. However their
works infers that one should exercise caution that the recess height is not too deep. This is
because the mass transport will completely change and target analytes can be trapped within
the recesses, allowing for atypical electrochemical responses consisting of convoluted
Faradaic and non-Faradaic contributions. Though their pioneering work was designed to
prove that electrochemistry and CE could be combined into one unit, they also used an array
of 100 electrodes that provided some temporal information about the chemical system, in
terms of monitoring changes in environment with respect to time, particularly in biological
cases. The internal heights and electrode separations were the focus of later work by the same
authors as they continued their quest to use amperometric platinum electrodes to detect
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dopamine using the same technology [7]. Their investigations proved that the platinum arrays
displayed a non-uniform sensitivity when detecting dopamine. This phenomenon is depicted
in Figure 2B, where it can be seen that a more concentrated injection of dopamine into the
chip results in a longer current depletion time than for a less concentrated injection. It is clear
that the two cases exhibit different sensitivities, thus complicated analysis would have to be
performed if the sample concentration was unknown. Furthermore, results were only valid
after a normalisation step that requires a pre-treatment stage prior to analysis, meaning that
though the concept is valid, it comes with some major practical limitations that require
addressing.
There were further reports about the integration of microelectrodes into
microchannels for in-stream electrochemical detection of target analytes shortly thereafter.
Darling et al. developed a microchip from silica containing etched channels via a wet
chemical process using EDP (a mixture of ethylene diamine and pyrocatechol) [8], reporting
on-board electrochemical detection via anodic striping voltammetry. Their work
demonstrated again that dopamine derivatives could be separated and detected using different
electrochemical methods on-board of a microchip. Work by Woolley et al. also utilised
similar chips to separate and detect neurotransmitters and DNA [9]. A pitfall to their work
was the separation voltage significantly interfered with the results and therefore relatively
small voltages had to be applied across the chip, meaning analysis times were longer than
desired. Yet this was proven to be offset by changing the working and reference electrode
spacing; the closer the detection electrodes were together, the less pronounced the
interference phenomenon. This is intuitive because the potentials in the detection area will
have less time to be influenced by the separation voltages. The gold working electrodes were
used in a single-electrode system, unlike previous cases that desired to create an
electropherogram typical of concentration or mass monitoring. The advantage to this method
is a simpler analysis procedure, but lacks the robustness of the array method because it
requires prior calibration and knowledge of analyte retention times under the specific
conditions monitored. Other electrodes were investigated by Wang and co-workers, such as
sputtered gold electrodes [10] and screen-printed carbon electrodes that were externally
mounted upon microchips [11]. The latter exhibited better sensitivities than previously
investigated on-board strategies. Later works furthered electrochemical detection in
microchips such as the report by Martin and co-workers regarding dual-electrode
electrochemical detection using Poly(DiMethylSiloxane), PDMS, fabricated microchips [12].
Their work was the first report of a dual-electrode microchip system, and also the first report
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of electrochemical detection using microchips fabricated from PDMS. At the time this was an
inherent advantage because chips could be fabricated from a master template repeatedly and
much quicker than glass chips that were etched using hydrofluoric acid. The dual-electrode
strategy allows for individual tailoring of working electrode surfaces to detect differing
components of a mixture, something that is near impossible on a single working electrode.
This is particularly useful if two compounds have similar m/z ratios, because they will reach
the detector at different times.
Unfortunately prior to 2005 there were very few reports of electrochemical detection
in ME and this was probably a limitation to the overall development of ME because many
detection methods such as LIF and MS are not portable, even if they exhibit enhanced
sensitivity and chemical reliability than a standard electrochemical method. However, the
early attempts opened up clear research pathways towards providing electrochemical
detectors. Some researchers opted for glass substrates that exhibit durability but lack the
speed of preparation that a PDMS substrate offers. Photolithography was generally employed
to apply platinum electrodes into such devices, yet gold electrodes were also suitable for
some applications, while sputtered and printed electrodes were also useful as disposable
options to be inserted into a chip, rather than built into the chip. The biggest problems were
with the separation voltage that is required for CE; a technical solution was offered by Gavin
and Ewing but is not used by others in this era. The potential for growth and development in
the field therefore hinges on chip design, electric field interference, electrode separation,
single/dual mode electrodes or arrays, fundamental understanding of diffusion regimes, and
finally the selectivity of the working electrodes, as it is clear that currents will be produced
for many biological species that undergo oxidation at noble metal surfaces under the
influence of electric potential. The field responded to these hurdles and many reports have
emerged since 2005 that have enhanced electrochemical detection in microchip technology.
The remainder of the review will focus upon more recent electrode materials for ME
applications.
Modern Devices
The boom in ME technology triggered a surge in academic literature regarding
electrochemical ME post-2005. Several reviews prior to this focussed upon electrochemical
detection, such as the contribution by Lacher et al., but though the work was novel then, the
electrodes described normally lacked the selectivity required for application in the field [13].
In particular, there was a focus upon standard electrode substrates such as gold, platinum, and
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glassy carbon, but with the evolution of electrochemistry, such standard substrates are seen
much less due to a lack of selectivity. Wang also provided reviews in 2002 and 2005, both of
which had a similar theme to the present review, but with a more self-fulfilling reference list
[1, 14]. There was a shift towards more selective ME devices utilising several electrode
substrates thereafter. A flurry of reviews focussing upon several defined aspects of
electrochemical ME was observed, such as Trojanowicz’s review of flow analysis strategies
[15], while others turned attention towards aptamers [16], and others take a general detection
direction [17]. This section encompasses electrode substrates and the engineering of
electrochemical microchips for specific applications, with a particular focus on dynamic
current-potential techniques such as voltammetry and amperometry.
Enzymatic Electrodes
A simplified philosophy of ME is to scale and laboratory techniques, combining the
injection, separation, and detection parts of an analytical method into one handheld unit. Thus
it is no surprise that the potential applications span forensic science, clinical chemistry, and
environmental science as these areas require on-the-spot analysis methods from complex
matrices. The first example of ME electrochemistry concerns the latter field. The monitoring
of phenolic compounds is a significant importance environmentally because they are harmful
to environmental systems and human health, yet many agricultural and petrochemicals still
contain phenols. Mayorga-Martinez et al. therefore designed a microfluidic chip with an
electrochemical detection port that selectively detects phenols [18]. Their screen-printed
carbon electrode design incorporates the use of tyrosinase, an enzyme that selectively reacts
with phenolic compounds, on board a composite designed to transduce enzymatic processes
into current transients. The benefits of the device depicted in Figure 3 is that the electrode can
be repeatedly replaced between measurements, meaning that cost effective screen-printed
electrodes offer a unique, cheap, and portable application for ME electrochemical detection.
The disposable screen-printed sensor, which transduces electrochemical reactions into
currents, simply has to be inserted into the device (Figure 3B) prior to use, and connected to a
potentiostat. Such disposable sensors cost the user a matter of pence. The modern potentiostat
is a handheld battery-powered device that can be transported to wherever required. In such an
application, the microchip and potentiostat can potentially be combined and transferred to the
site of an environmental sample, reducing sample contamination. A combination of CE and
electrochemistry is required for such samples in order to separate the analyte from the matrix
first so that electrochemical interference from background analytes is reduced.
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Application of ME to the clinical industry is just as important as to the environment.
One example is that of the hydrogen peroxide sensor designed by Matharu and co-workers
[19], which is cleverly utilised to understand how specific cell cultures interact with alcohol.
The ME detection system, depicted in Figure 4, is fabricated in a multi-step system that
requires several components in order to successfully transduce chemical interaction events
into current signals. A gold electrode is modified with silane, polyethylene glycol, and
HorseRadish Peroxidase (HRP). The saline surrounding the electrode in Figure 4 serves as a
bed for hepatocytes, which are liver cells responsible for detoxification of blood. When
alcohol is injected into the device, the hepatocytes respond by breaking the alcohol down into
hydrogen peroxide and acetal, in a 2 electron 2 proton process that takes place at the cell
membrane surfaces. The liberated extracellular hydrogen peroxide is broken down by the
HRP fixed on the gold electrode surface and transduced into a current signal. The array of
modified electrodes utilised allows for a time-dependent electropherogram that could be used
to study the rate of alcohol degradation, and also the introduction of antioxidants is studied to
observe the effect of antioxidants on peroxide formation and oxidative stress levels.
Within the last three years there have been many examples of enzyme-based
electrochemical microfluidic devices that are worth mentioning. Instead of discussing each
application individually, Table 1 has been provided for interested readers to view different
enzymatic detection strategies for a range of applications including cholesterol, HIV, and
glucose [20-24].
Graphene-Based Electrodes
In line with modern electrochemistry, graphene-based electrodes have also been
exhaustively investigated for application in ME devices. One such report was by Chua and
Pumera, who focussed upon reduced graphene oxides for use as detection electrodes for
dopamine and catechol [25]. These electrodes are created by suspending lab-synthesized
graphenes in an organic solvent, which is drop-casted upon a glassy carbon electrode using a
standard Eppendorf pipette. The reduced graphenes (thermally, chemically, and
electrochemically reduced) displayed no inherent advantage over normal carbon electrodes
such as glassy carbon for the detection of dopamine and catechol in such systems. The
authors attribute this to an increase in background current exhibited by the electrode
composites, yet it is likely that the increased basal nature of graphene compared to other
graphitic materials is responsible for poor peak currents. Furthermore, the electrode is
essentially being utilised as a channel flow electrode in such cases, so the constant laminar
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flow will have a detrimental impact upon the drop-casted surface by mechanically stripping
the composite from the surface, due to no covalent linkage of the graphene to the substrate.
Solution-based graphenes are also utilised to detect cholesterol by Ruecha and co-
workers [26]. Their method uses the conducting properties of graphene to improve the
conductivity of a polymer electrode. The graphenes were mixed with polyvinylpyrrolidone
and polyaniline, creating graphene-infused nanostructures that can be electrosprayed upon a
substrate. In their work, the mixture is electrosprayed upon paper with cholesterol oxidase,
thus cholesterol is broken down, liberating hydrogen peroxide as a biproduct that is detected
amperometrically, similar to many other cases. The utility of graphene in this example is to
make a conducting polymer that can be sprayed easily onto a ME device, thus creating
sensitive and selective electrodes that are easy to mould onto the desired substrate.
Carbohydrates are also useful to detect and often appear in complicated matrices.
Thus, work by Zhang et al. focussed upon fabricating graphene and nickel nanoparticles that
could be grafted upon resin microspheres for the detection of specific carbohydrates in ME
systems [27]. Their work exhibited a sensitivity towards glucose, sucrose, and fructose of
200-300 nA mM-1
using an amperometric detection method with a pipette tip as an electrode
case. Further examples of graphene-based microfluidic working electrodes are given in Table
2 [28-32].
Carbon Nanotube-Based Electrodes
Drop-casting of carbon nanomaterials upon substrates is by no means limited to
graphenes. In fact, Carbon NanoTubes (CNTs) were fabricated and studied at length long
before graphenes ever were and are still researched today. CNTs such as Single-Walled and
Multi-Walled CNTs (MWCNT/SWCNT) are generally utilised by researchers to reduce
activation potentials for electrochemical processes, thus making electrodes more efficient,
stable, and longer lasting. CNTs allow this because their anisotropic structures orient
themselves in a manner in which the electronically concentrated zones are exposed as the
electrode surface [33]. This is the opposite phenomenon to graphene, where the structures
orient themselves so that the electronically sparse basal planes are exposed as the electrode
surface. The advantageous electrochemistry of CNTs has therefore attracted much attention
for electrodes in ME devices.
One such example of an integrated CNT electrode on a microchip is by Xu and co-
workers, who incorporate CNTs into a polystyrene matrix in order to fabricate a conducting
polymer electrode, which can be applied for the detection of active ingredients in herbal
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medicines [34]. The polymer is mounted upon a copper wire and cased within a silica tube to
define a working electrode with a finite diameter of 2 mm. The fabricated electrode was
inserted into a chip designed to accommodate an electrode of such a diameter. The CNT
composite provided a reduced activation potential for rutin of +0.49 V, compared to +0.67 V
when compared to a graphite composite of the same design. This reduction in peak potential
is a result of the electronic anisotropy as mentioned previously, and allows a lower energy
activation in the chip that is advantageous in terms of peak resolution. These in-situ
approaches are commonplace for microchip electrode design. Other works report SWCNT
electrodes obtained by press-transfer technology on PMMA substrates for ME applications
[35]. Through careful optimisation of the conditions, the press-transferred electrodes can
exhibit excellent repeatability, an extreme resistance to fouling (unlike in the case of
graphenes), remarkable signal-to-noise characteristics, and a well-defined linear
concentration dependence. Other works using CNTs are provided in Table 3 [36-41].
Screen-Printed Electrodes
Modern research focusses upon many substrates for detection of target species, yet
sometimes the simplest technologies are the most useful. Screen-printed carbon electrodes,
sometimes referred to as thick-film electrodes [1], exhibit a host of advantages over thin-film
electrodes and conventional electrodes such as glassy carbon and gold. They are cheap, mass
producible, disposable electrodes that often require no recourse or pre-treatment steps. They
are far easier to fabricate than thin-film electrodes, are more reliable in terms of mechanical
stress, and require simple and relatively inexpensive old printing technologies instead of
deposition techniques such as physical vapour deposition or chemical vapour deposition. It is
for these reasons that screen-printed technologies find uses in many areas including clinical
[42, 43], forensic [44-46], and environmental science [47].
In terms of microchips, there have already been examples described above that utilise
screen-printed electrodes in microchip detectors. One such example is that of the Prussian-
blue modified screen-printed electrode that is utilised to detect glucose, as reported by Sekar
and co-workers [48]. A paper-based substrate was utilised in this example as an ideal matrix
to house glucose oxidase, providing the specificity for glucose. The Prussian blue is
incorporated due to its ability to reduce hydrogen peroxide at a lower potential, thus allowing
the sensor to be operated using low energies that will not disturb other chemical constituents
of a sample matrix.
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Of course, it would be better to create a detection system that was free from enzymes
because the electrode would not be limited by the strict operating conditions that are required
to maximise enzymatic interactions. Unfortunately most examples are restricted to enzyme-
on-paper coupled with enzymeless electrochemical detection. Yang and co-workers employ a
novel screen-printed system for microfluidic usage using such a system, where platinum
nanoparticles act as the sensor for hydrogen peroxide on a screen-printed electrode [49]. A
specific sensitivity of 10 µA mM-1
cm-2
indicates a very useful sensing platform for hydrogen
peroxide and may be the superior choice of electrode for any case that uses enzymatic
processes as the majority create hydrogen peroxide as its primary biproduct. However this
strategy may not be suitable for devices wishing to exhibit more than a few uses, because
hydrogen peroxide is known to foul substrates quite corrosively. In terms of a disposable one
shot sensor, these strategies are amenable, however.
Laser printing is also a viable option, as demonstrated in work by Coltro et al [50].
Such devices used laser printed electrodes for the detection part of the ME device, a strategy
that is quite simple in principle. Their device was demonstrated to detect iodide and ascorbate
to levels as low as 500 and 1800 nmol L-1
, respectively. There also exist other approaches,
such as the use of copper nanowires as electrode materials for the ME sensing of
galactosemia [51], or a similar approach for the determination of saccharides in honey [52],
highlighting the seemingly endless possibilities that electrode design can offer for ME
applications. Microchip electrophoresis–copper nanowires for fast and reliable
determination of monossacharides in honey samples
There are simply too many examples of electrodes to surmise in one review alone.
This review as tried to capture some of the electrodes that can be incorporated directly into
ME, yet other reviews have found many more avenues to explore. For example, gold
nanoparticles are utilised by some researchers in DNA applications [53]. A review by Pumera
summarises how ME can be utilised for the detection of explosives [54], while Matysik’s
review focusses upon the advances of amperometry within ME applications [55]. Dungchai’s
review also looks at electrochemical detection strategies, this time in paper-based devices [3].
Finally, Xu et al focus their review upon different electrochemical detection modes utilised
within ME [56].
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Concluding Remarks
A range of electrochemical detection strategies are available that are feasible on the
micro-scale and have been successfully applied to ME. From old electrodes such as gold and
platinum, these cases supply the advantage of good sensitivities, but with the compromise of
a complicated production procedure such as lithography and they come at a good deal of
expense. Thus other strategies such as, enzymes, printed carbons, and graphenes were
focussed upon by the field in order to reduce electrode size, cost, improve reproducibility and
be mass produced, while maintaining a level of selectivity for specified applications. Many
electrodes succeed in this endeavour and create strategies that may be suitable for such
applications, but others less so. Solution-based graphenes are unreliable due to weak non-
covalent linkages to electrode substrates allowing mechanical stripping under high voltage
laminar flow. Enzymatic technologies are selective but require specific conditions and
storage that may limit their practical application in the long term. Screen-printed electrodes
hold an advantage of being mass-producible but require specific tailoring of surfaces in order
to detect molecules selectively. As seen within this short review, researchers tend to opt for a
combination of different routes to tailor the electrode response, most of which utilise the
hydrogen peroxide pathway and a chronoamperometric detection method. There are
questions over the long term suitability of this given that hydrogen peroxide forms radicals
and attacks many different substrates, but a continuation of this philosophy is likely because
such electrodes give the highest selectivity in a given application. The improvement of
electrochemical detection methods will continue and will likely form the first choice method
for ME detection in the coming years.
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Figure 1 Schematic diagram of the microfluidic creatinine device in Reference [4]. The
different components are as follows: (1) sample opening with applied sample droplet; (2)
evaporation reservoir; (3) injection channel for injection of cations by moving boundary
electrophoresis; (4) double-T injector; (5) reservoir with gas bubble for liquid expansion
control; (6) conductivity detection electrodes; (A and B) high-voltage injection anode and
cathode; (C and D) high-voltage separation anode and cathode; (E) electrodes for the
determination of the sample conductivity.
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Figure 2 Different analyte concentrations monitored with the microfabricated
electrophoresis-electrochemical array detection scheme. (A) Plugs of dopamine (0.665 or
3.46 mM; 45 s duration) were injected from a sample vial into a fused silica capillary (10 µm
internal diameter, 70 cm length) that was coupled to a buffer-filled rectangular channel (8 µm
internal height, 4.8 cm length). The capillary and channel were filled with MES buffer (25
mM, pH 5.9) prior to the start of the experiment. The capillary and channel voltages used
were 28 kV and 1500 V, respectively. The capillary was moved at a rate of 0.2 s/step. The
separation current was 0.19 mA. Detection was at +0.8 V vs. Ag/AgCl. (B) Three-
dimensional plot of the electropherogram shown in (A). The three-dimensional plot displays
changes in current depletion with respect to concentration of the injected analyte, where the
spikes on the left represent the higher concentration (slower current depletion) and the spikes
on the right represent the lower concentration (faster current depletion). Reprinted from
Reference [7] with permission from the American Chemical Society.
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Figure 3 Schematic representation of the PDMS/glass microchip device components (A).
Photograph of the PDMS/glass fluidic microsystem setup (B). Schematic representation of
the microchip design with the channel and SPE modified with CaCO3-poly(ethyleneimine)
(PEI) microparticles (MPs) and tyrosinase (C). Reproduced from Reference [18] with
permission from Wiley.
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Figure 4 Steps 1−3. Fabrication of enzymatic microstructures on gold electrodes. Steps 5−6.
Culture and injury of hepatocytes followed by electrochemical detection of extracellular
peroxide. Reprinted from Reference [19] with permission from the American Chemical
Society.
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Table 1 Electrochemical microchip technologies employing enzymatic detection methods.
Electrode Application Sensitivity Comments Ref.
1. Multi-walled carbon
nanotubes
2. Nickel oxide nanoparticles
3. Cholestrol oxidase and
cholesterol esterase
Cholesterol 2.2 mA mM-1
cm-2
Use of covalent linkages
between terminating species on
nanotubes connect enzyme to
the substrate
[20]
1. Acetylcholinesterase upon
gold
2. Silver
Thiocholine 0.597 µA mM-1
Selective amino acid
determination
[21]
1. Gold
2. Magnetic beads
3. HIV antibodies
HIV N/A Impedimetric sensor relies upon
non-Faradaic interactions,
producing current transients
similar to antibody/antigen
interactions
[22]
1. Screen-printed carbon
electrode
2. Lactate oxidase
Lactate 0.317 µA mM-1
Amperometric measurement
detects hydrogen peroxide
produced from the reaction of
lactate oxidase with lactate from
saliva on a cotton-based
substrate
[23]
1. Platinum
2. Platinum black
3. Glucose oxidase
Glucose ca. 0.25 µA mM-1
Platinum black improves surface
area for signal transduction
[24]
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Table 2 Electrochemical microchip technologies employing graphene-based sensing
electrodes.
Graphene Application Comments Ref.
Single-layer micropatterned
graphene nanohybrids
HIV antibodies Current response indicative of HIV
antibodies
[28]
Polyaniline-wrapped
graphene
Electro-osmotic pump Non-sensing application but worthy
of a mention due to its innovation
[29]
Reduced graphene oxide Cancer biomarkers Four cancner biomarker sensing
routes developed using the same
paper-based methodology
[30]
Graphene and gold Mercury 10 ppm detection limit for
environmental sensing
[31]
Graphene-modified gold
paper electrode
DNA hybridization Detect DNA in femtomolar levels [32]
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Table 3 Electrochemical microchip technologies employing CNT-based sensing electrodes.
CNT Application Comments Ref.
Press-transferred SWCNT Isoflavones 250 s response time with high accuracy [37]
CNTs for in-channel flow
enzyme immobilization
Cholesterol Low cross sensitivity towards interfering
species
[38]
SWCNT/MWCNT Dopamine Qualitative study displays the potential
electrocatalytic properties of CNTs
[40]
Copper nanocluster and
SWCNT electrode
Glucose Linear range spans four orders of
magnitude
[39]
MWCNT and cobalt
hexacyanoferrate
Hydrazine Useful for environmental monitoring [41]